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1.
The fusion protein formed from ch14.18 and interleukin-2 (ch14.18-IL-2), shown to exhibit antitumor efficacy in mouse models, consists of IL-2 genetically linked to each heavy chain of the ch14.18 chimeric anti-GD2 monoclonal antibody. The purpose of this study was to determine the pharmacokinetics of ch14.18-IL-2 in mice and assess its stability in murine serum. Following i.v. injection, the fusion protein was found to have a terminal half-life of 4.1 h. Detection of IL-2 following injection of the ch14.18-IL-2 fusion protein showed a similar half-life, indicating that the fusion protein prolongs the circulatory half-life of IL-2. Detection of human IgG1 following injection of ch14.18-IL-2 showed a terminal half-life of 26.9 h. These data suggested that the native fusion protein is being altered in vivo, resulting in a somewhat rapid loss of detectable IL-2, despite prolonged circulation of its immunoglobulin components. In vitro incubation of the ch14.18-IL-2 fusion protein in pooled mouse serum at 37 degrees C for 48 h resulted in a loss of its IL-2 component, as detected in enzyme-linked immunosorbent assay systems and in proliferation assays. Polyacrylamide gel electrophoresis and Western blot analysis of the fusion protein incubated in mouse serum at 37 degrees C indicated that the ch14.18-IL-2 is cleaved, resulting in a loss of the 67-kDa band (representing the IL-2 linked to the IgG1 heavy chain) and the detection of a band of more than 50 kDa, slightly heavier than the IgG1 heavy chain itself. This suggests that the fusion protein is being cleaved in vitro within the IL-2 portion of the molecule. These studies show that (1) ch14.18-IL-2 prolongs the circulatory half-life of IL-2 (compared to that of soluble IL-2) and (2) the in vivo clearance of the fusion protein occurs more rapidly than the clearance of the ch14.18 antibody itself, possibly reflecting in vivo cleavage within the IL-2 portion of the molecule, resulting in loss of IL-2 activity.  相似文献   

2.
The C6.5 endoglucanase from Bacillus subtilis catalyzes the hydrolyses of beta-glucans. This enzyme, which is also produced by many ruminant microbes, is not part of the normal digestive repertoire of monogastric animals. We have generated transgenic mice which express the C6.5 endoglucanase gene specifically in the pancreas with secretion of the enzyme into the small intestine. The secreted enzyme has a molecular mass of 55 kDa which is reduced by protease digestion to the principal forms of 37 and 35 kDa. These truncated forms are resistant to further protease degradation and exhibit enhanced specific activity compared to the native enzyme. These results encourage further investigation of the utility of this transgene for enhancing the digestive capability of monogastric animals.  相似文献   

3.
Interleukin 5 (IL-5) is a kind of peptide hormone released from T lymphocytes of mammals infected with microorganisms or parasites. It is an acidic glycoprotein with a molecular mass of 40 to 50 kDa that consists of a homodimer of polypeptides. It controls hematopoiesis so that it increases natural immunity. In the mouse, IL-5 acts on committed B cells to induce differentiation into Ig-producing cells and on common progenitors for CD5+ pre-B cells and CD5+ macrophages to support their survival. The antibodies secreted by CD5+ B cells seem to be responsible for the primary protection against the infection with microorganisms or parasites. It also supports the growth and/or differentiation of eosinophil precursor and mature eosinophils, which can be effective for the removal of parasites in combination with the antibodies against them. Murine IL-5 receptor (IL-5R) consists of two different polypeptide chains; alpha chain and beta chain. The IL-5R alpha chain is 60 kDa protein that binds IL-5 with low affinity. The IL-5R beta chain is a 130 kDa protein which does not bind IL-5 by itself but is necessary to form the high affinity IL-5R. The beta chain was identified by using one of the anti-IL-5R mAb and anti-IL-3R mAb as the IL-3R homologue. This beta chain is also used as the beta chain of GM-CSF receptor. This fact suggests that there is a common signaling mechanism among these cytokines and efficient cooperation among them. At the same time, these findings may explain the overlapping role of these cytokines in the development of granulocytes.  相似文献   

4.
This study characterised a 90 kDa lectin from an invertebrate chordate, the tunicate Styela plicata. One- and two-dimensional electrophoresis showed that the apparent molecular weight of this protein is maintained under both reducing and non-reducing conditions, suggesting that its native form is a monomer. The 90 kDa lectin was localised within a single type of hemocyte (morula cells), but was secreted from those cells when tunicates were challenged with the inflammatory elicitor, zymosan. Functional studies showed that the 90 kDa protein binds to galactose-based sugars in a divalent cation-dependent manner. Amino acid composition analysis and N-terminal amino acid sequencing indicated that the 90 kDa lectin is related to a previously characterised, collagenous lectin from S. plicata, splic43. However, peptide mass fingerprinting identified numerous differences between the two proteins. This suggests that the 90 kDa molecule represents a novel protein that is involved in host defence.  相似文献   

5.
SPC1 (furin/PACE), an enzyme belonging to the S8 group of serine endoproteases, is a type I integral membrane protein that catalyzes the processing of a multitude of precursor proteins. We report here the use of transfected Drosophila melanogaster Schneider 2 cells to produce milligram amounts of two forms of recombinant human SPC1. In order to investigate the role of the cysteine-rich region (CRR) of SPC1, we compared the biochemical and enzymatic properties of hSPC1/714 that has the C-terminal tail and transmembrane region of the native enzyme removed with that of hSPC1/585 which had, in addition, the CRR deleted. Two stable cell lines were established. The S2-hSPC1/714 line secreted a major form of apparent molecular weight of 83 kDa and a minor form of 80 kDa whereas the S2-hSPC1/585 line secreted a single 59-kDa protein. PNGase F treatment of the different forms demonstrated that the enzymes were glycosylated. Automated NH(2)-terminal sequencing revealed that all purified forms resulted from processing at the expected zymogen activation site. Removal of the CRR resulted in a broadening of the enzyme's pH range, a shift of K(0.5) for Ca(2+), and a shorter enzymatic half-life when compared to the longer form, which suggest that the CRR of hSPC1 may help in stabilizing the enzyme's proteolytic activity. The use of this high-level expression system will meet the demand for material necessary to perform biochemical and structural studies that are needed to further our understanding of this and other SPCs at the molecular level.  相似文献   

6.
The sensitivity of the membrane-bound hydrogenase of Bradyrhizobium japonicum to inactivation by proteases and membrane-impermeant protein modification reagents was compared under hydrogen versus oxygen. In membrane vesicles, the half-life of enzyme inactivation by trypsin of the H2-reduced enzyme was approximately 10 min, whereas O2-oxidized enzyme was much less sensitive to trypsin inactivation (half-life of over 90 min). Diazobenzene sulfonate (DABS) affected the enzyme activity in a manner similar to proteases. With DABS, the enzyme had a half-life of 2-3 min under H2 versus over 30 min under O2. Experiments in which the gas phase (containing either H2 or O2) available to the membranes was changed prior to the protease or chemical modification treatments indicated that it is the redox state of the enzyme at the time of the treatment which determines the sensitivity of the enzyme to inactivation. The redox-dependent differences in the behavior of the membrane-bound enzyme were attributed to changes in the accessibility of the small (33 kDa) subunit. The kinetics of enzyme inactivation by trypsin, under H2, correlated very well with the degradation of the intact 33-kDa subunit, whereas the large subunit (65 kDa) was rather resistant to proteolytic degradation. DABS treatment was found to decrease the reactivity of the small subunit to its antibody concomitant with enzyme inactivation under H2, but without such an effect on the O2-oxidized enzyme. In contrast to the results with the membrane-bound enzyme, purified dehydrogenase was found to be equally susceptible to inactivation by proteolysis or chemical modification irrespective of whether the treatments were performed under H2 or O2. These results indicate that, in the membrane, hydrogenase undergoes a redox-linked conformational change, whereby the small subunit of the enzyme becomes more accessible to external reagents when the enzyme is in its reduced form.  相似文献   

7.
Acetolactate synthase (ALS, AHAS; EC 4. 1. 3. 18) from Brassica napus has been partially purified and characterized using polyclonal antibodies. Following denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blot analysis, 65 and 66 kDa ALS subunit polypeptides were immunologically detected, along with a novel 36 kDa polypeptide which cross-reacted with the anti-ALS antibody. Partial peptide sequencing of the 36 kDa peptide revealed significant similarity to plant aldolase proteins. ALS activity from stromal extracts fractionated by gel filtration chromatography as a single species of estimated molecular mass of 124 kDa, while comparative sedimentation coefficient in glycerol gradients indicated a corresponding molecular mass of 132 kDa. The results suggest that the native enzyme is a dimer of 65 and/or 66 kDa subunits. Anion exchange chromatography resolved two classes of ALS activity of equal native molecular weight, but which exhibited different properties with respect to subunit structure, sensitivity to inhibition by chlorsulfuron and feedback inhibition by branched chain amino acids.  相似文献   

8.
Gaussia分泌型萤光素酶是近年发现的一种来源于海洋桡脚类动物Gaussia princeps的新型分泌型萤光素酶,是目前已知的可自然分泌的最小萤光素酶,因其分子小、灵敏度高、半衰期短和可高效分泌,而成为一种理想的报告基因,广泛应用于体内外研究。我们就Gaussia分泌型萤光素酶的发光原理、荧光特性及其应用等进行简要综述。  相似文献   

9.
Tissue-type plasminogen activator (t-PA) is a mosaic protein containing several distinct structural domains attached to the serine protease catalytic unit present at its COOH terminus. To investigate structure-function relationships in t-PA, we deleted the NH2-terminal domains, finger and epidermal growth factor, by genetic engineering. The genes for the parent and mutant t-PA were expressed in a bovine papilloma virus-dependent mammalian cell system. The secreted proteins were purified to homogeneity. The mutant protein was processed to the expected size of about 60 kDa compared to approximately 68 kDa for the parent t-PA, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography. While the mutant t-PA had amidolytic activity comparable to native t-PA, it did not bind appreciably to fibrin. Consequently, fibrin-dependent enzymic activity, i.e. plasminogen activation in the presence of soluble fibrin and fibrinolysis were lower than with native recombinant t-PA. The effect of deletion of NH2-terminal domains on the plasma half-life (t1/2) was investigated by injecting native and mutant t-PA into mice. While the majority of the t-PA disappeared initially with a t1/2 of about 2 min, mutant t-PA cleared at a much slower rate with t1/2 of about 50 min. These findings suggest that the NH2-terminal domains of t-PA not only determine its specificity for binding to fibrin but also mediate its clearance from plasma in vivo. Furthermore, the catalytic unit in t-PA seems to function autonomously.  相似文献   

10.
IL-7 induction of protein tyrosine phosphorylation was examined in an IL-7-dependent thymocyte cell line, D1, which was generated from a p53-/- mouse. Anti-phosphotyrosine antibody was used both to immunoprecipitate and Western blot, and showed that IL-7 induced tyrosine phosphorylation of a protein with a molecular weight of approximately 200 kDa. The P200 band was purified by reversed-phase high-performance liquid chromatography. Amino acid sequencing by mass spectrometry revealed three peptides identical to rat clathrin heavy chain (CHC) 1 (192 kDa), and this was confirmed by blotting with an anti-clathrin antibody. Stimulation of normal pro-T cells by IL-7 showed an increased tyrosine phosphorylation of clathrin heavy chain. Tyrosine phosphorylation of clathrin heavy chain was strongly induced by IL-7 and to a lesser extent by IL-4, while no effect could be observed with the cytokines IL-2, IL-9 and IL-15, whose receptors share the gammac chain. Phosphorylation of clathrin heavy chain was found to be sensitive to Jak3 inhibitors but not to Src inhibitors. Clathrin is involved in internalization of many receptors, and its phosphorylation by IL-7 stimulation may affect the internalization of the IL-7 receptor.  相似文献   

11.
We have identified a new factor, CFX, in human serum and plasma that inhibits the growth of cultured human and mouse cell lines. CFX was determined to be a negatively charged, hydrophobic glycoprotein, with a native molecular weight of 110–120 kDa and a minimal active subunit of 55 kDa. It is precipitated by 60% ammonium sulfate and is resistant to heat treatment at 100°C for 30 min. CFX was purified from human plasma to a single band on a gel which retained the cell growth inhibitory activity. Amino acid sequence analysis of the CFX band revealed sequences from four human glycoproteins, α1-antichymotrypsin, C1-esterase inhibitor, α1-antitrypsin, and α2-antiplasmin, all members of the superfamily of serpins. Of the four, C1-esterase inhibitor was shown to be the most potent cell growth inhibitor. These results suggest that serpins may play a cell growth inhibitory role in vivo, in addition to their role as protease inhibitors.  相似文献   

12.
The kidney bean lectin Phaseolus vulgaris phytohemagglutinin E-form (PHA-E) was expressed and secreted by the methylotrophic yeast Pichia pastoris. To optimise yields of PHA-E, transformants of P. pastoris were selected for high-level production of the recombinant protein. A scaleable process for the production and purification of gram quantities of recombinant PHA-E is reported. PHA-E was secreted at approximately 100 mg/L at the 2- and 200-L scale and was purified to 95% homogeneity in a single step using cation-exchange chromatography. The purified recombinant PHA-E consists of four forms with molecular masses between 28.5 and 31.5 kDa, as assessed by MALDI-TOF, whereas its native counterpart has a molecular mass of approximately 30.5 kDa. Endoglycosidase treatment revealed that the range in size of the recombinant protein was attributed to differences in the nature of the N-linked oligosaccharides bound to the protein. The primary amino acid sequence of the recombinant PHA-E was found to be identical to the native protein and to have an agglutination activity similar to that of native PHA-E. The data presented here suggest that, using P. pastoris, gram quantities of a recombinant phytohemagglutinin E-form can be produced and that the recombinant protein is similar to the protein synthesised in plants with respect to structure and biological activity.  相似文献   

13.
Mammalian IL-1beta is produced as a biologically inactive 31 kDa precursor, which is converted to the active 18 kDa form by proteolytic processing. Synthesis and processing of native piscine IL-1beta is poorly understood. In the present study, the native IL-1beta precursor or mature peptides were detected at sizes of approx. 29 kDa and 24 kDa in cell lysates of a rainbow trout macrophage cell line RTS-11, with or without LPS stimulation, by Western blot analysis using a polyclonal antibody against the putative trout mature IL-1beta (rmIL-1beta) produced in Escherichia coli. Processing of the 29 kDa precursor into a 24 kDa mature peptide was confirmed by analysis of such proteins using a monoclonal conjugate (Ni-NTA-HRP) against 6 histidines in lysates of the RTS-11 cells transfected with an expression plasmid containing the IL-1beta precursor molecule tagged with 6 histidines at its C terminus. Only the recombinant mature 24 kDa) IL-1beta/HIS protein was purified from the culture supernatants of the transfected cells, indicating the molecule is cleaved to be secreted. These findings strongly suggest that the trout IL-1beta molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1beta despite the lack of a clear ICE cut site.  相似文献   

14.
Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas comosus) and is unique in containing a single oligosaccharide chain attached to the polypeptide. This property allowed its affinity binding and favorable orientation on a Sepharose support pre-coupled with the lectin, concanavalin A (Con A). For comparison, bromelain was also immobilized by covalently coupling to the CNBr-activated Sepharose. The preparation obtained was more resistant to thermal inactivation as evident from the retention of over 50% activity after incubation at 60 for 100 min (as compared to 20% retained by the native enzyme and 30% retained by the covalently immobilized enzyme), exhibited a broader pH-activity profile with the enzyme retaining over 60% activity at pH 11 (as compared to over 25% retained by native and the enzyme immobilized covalently). The native, covalently-coupled and affinity-bound bromelains had apparent K m values of 1.1, 2 and 0.54 mg/ml, respectively using casein as the substrate. The V max values remained unaffected on immobilization.  相似文献   

15.
The enzyme consists of a single polypeptide chain of molecular weight of 64,000 and tends to form heavy aggregates. It is sensitive to rifampicin and resistant to α-amanitin. It prefers native mitochondrial DNA and d(AT) copolymer as template.  相似文献   

16.
Inhibition of human blood coagulation factor XIa by C-1 inhibitor   总被引:1,自引:0,他引:1  
The inactivation of activated factor XI (factor XIa) and of its isolated light chain by C-1 inhibitor was studied. Irreversible inhibition was observed in a reaction in which no reversible enzyme-inhibitor complex was formed. The second-order rate constants for the inactivation of factor XIa or its light chain by C-1 inhibitor were 2.3 X 10(3) and 2.7 X 10(3) M-1 s-1, respectively. High molecular weight kininogen did not affect the rate of inactivation. The nature of the complexes formed between factor XIa or its light chain and C-1 inhibitor was studied by using sodium dodecyl sulfate gradient polyacrylamide slab gel electrophoresis. Under nonreducing conditions, two factor XIa-C-1 inhibitor complexes were observed with apparent molecular weights of 230,000 and 300,000. Reduction of these complexes resulted in the formation of a single band with a molecular weight of 130,000. This band is also formed in the reaction of the isolated light chain of factor XIa with C-1 inhibitor. These results demonstrate that two C-1 inhibitor molecules can become bound to the light chains of a factor XIa molecule. In addition, the mechanism of interaction of factor XIa or its isolated light chain with C-1 inhibitor appears identical, and the rate of inactivation of the enzyme by C-1 inhibitor is very similar. Neither the heavy chain of factor XIa nor high molecular weight kininogen is significantly involved in the inactivation of factor XIa by C-1 inhibitor.  相似文献   

17.
Different tyrosines are autophosphorylated on the native and on the protease-generated 150 kDa forms of the epidermal growth factor receptor. High ATP concentrations increase the apparent molecular weight of already phosphorylated native receptors but not of the 150 kDa form, indicating that only the native receptor has multiple autophosphorylation sites available. The non-identity of the tyrosine-phosphates on the native and 150 kDa receptor forms is seen in their response to alkaline hydrolysis (10% and 40% resistant, respectively). Since the liberated phosphate is peptide bound, the native receptor fails to be alkali-resistant because of which peptide bonds are hydrolyzed.  相似文献   

18.
Arginase was purified from Vigna catjang cotyledons and buffalo liver by chromatographic separations using Bio-Gel P-150, DEAE-cellulose and arginine AH Sepharose 4B affinity columns. The native molecular weight of an enzyme estimated on Bio-Gel P-300 column for Vigna catjang was 210 kDa and 120 kDa of buffalo liver, while SDS-PAGE showed a single band of molecular weight 52 kDa for cotyledon and 43 kDa for buffalo liver arginase. The kinetic properties determined for the purified cotyledon and liver arginase showed an optimum pH of 10.0 and pH 9.2 respectively. Optimal cofactor Mn++ ion concentration was found to be 0.6 mM for cotyledon and 2 mM for liver arginase. The Michaelis-Menten constant for cotyledon arginase and hepatic arginase were found to be 42 mM and 2 mM respectively. The activity of guanidino compounds as alternate substrates for Vigna catjang cotyledon and buffalo liver arginase is critically dependent on the length of the amino acid side chain and the number of carbon atoms. In addition to L-arginine cotyledon arginase showed substrate specificity towards agmatine and L-canavanine, whereas the liver arginase showed substrate specificity towards only L-canavanine.  相似文献   

19.
A human neuroblastoma cell line (Paju) grew in 10 mM difluoromethyl-ornithine, which at this concentration normally stops the growth of all mammalian cells. Ornithine decarboxylase from Paju was resistant to inhibition in vitro by difluoromethylornithine, and required 10 microM of the compound for 50% inhibition, whereas ornithine decarboxylase from SH-SY5Y cells (another human neuroblastoma) and from rat liver needed only 0.5 microM difluoromethylornithine. Paju ornithine decarboxylase also exhibited a long half-life (over eight hours) in vivo. The half-life of immunoreactive protein was significantly longer than that of the activity. The long half-life of ornithine decarboxylase in Paju cells leads to its accumulation to a specific activity of 2000 nmol/mg of protein per 30 min during rapid growth (the corresponding activity in SH-SY5Y cells was about 2.5). When partially purified ornithine decarboxylase from Paju cells was incubated with rat liver microsomes it was inactivated with a half-life of 75 min. This inactivation was accompanied by a fall in the amount of immunoreactive protein. In the same inactivating system partially purified SH-SY5Y ornithine decarboxylase had a half-life of 38 min and its half-life in vivo was 50 min. The corresponding values for rat liver ornithine decarboxylase were 45 min and 40 min, respectively. Rat liver microsomes also inactivated rat liver adenosylmethionine decarboxylase. These results suggest that Paju ornithine decarboxylase has an altered molecular conformation, rendering it resistant to (i) difluoromethylornithine and (ii) proteolytic degradation both in vivo and in vitro.  相似文献   

20.
By use of immunoblot analysis, we demonstrate the presence of a pro-Epidermal growth factor (EGF) with an approximate molecular weight of 165 kDa in adult mouse urine. In addition, urine contains four high molecular weight EGFs with approximate molecular weights of 116, 97, 66 and 56 kDa. The 165 kDa pro-EGF as well as the 66 and 56 kDa EGFs also are detectable in mouse kidney extract. Neither urine nor kidney contain the mature EGF of 6 kDa. The 165 kDa pro-EGF is the major product synthesized in renal tissue and secreted in urine. The finding of high molecular weight EGFs in urine suggests that part of pro-EGF secreted into urine undergoes partial proteolysis distal to its site of synthesis.  相似文献   

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